Siglec inhibitors

ABSTRACT

The invention relates to Siglec inhibitors that have an increased affinity for the receptor molecule. The Siglec inhibitors provided by the invention are preferably selective of a given Siglec molecule. The invention further relates to a method for producing Siglec inhibitors and to a method for increasing the binding selectivity for a given Siglec molecule. The invention also relates to pharmaceutical compositions that contain the Siglec inhibitors and to medical indications for the Siglec inhibitors.

This application is the U.S. national phase of international patentapplication PCT/EP02/06277, filed on Jun. 7, 2002, and claiming priorityto German patent application nos. 10129332.1, filed Jun. 19, 2001, and10216310.3, filed Apr. 12, 2002, all of which are hereby incorporated byreference.

The invention relates to siglec inhibitors and pharmaceuticalcompositions which contain them. Furthermore, the invention relates tomethods of increasing the binding selectivity of siglec inhibitors andspecification of the medical indications of the siglec inhibitorsprovided.

Siglecs (sialic acid binding Ig-like lectins) are Ig-type lectins whichare characterised by an N-terminal V-set domain which mediates thesialic acid bond. A varying number of Ig domains of the C2 set followsthe Ig domain. Originally, the lectin family was found based onindependent studies of sialoadhesin (siglec-1 CD 169), a macrophagelectin-like adhesion molecule and CD22 (siglec-2), a B-cell restrictedmember of the Ig superfamily (IgSF), which plays an important role inthe regulation of the B-cell activation. It was also found that bothmolecules mediate the cell-cell interactions in vitro by the detectionof sialylated glycoconjugates. The cloning of sialoadhesin indicatedhigh sequence similarities to CD22 and led to the conclusion that twofurther IgSF proteins, having a relationship in this respect, themyelin-associated glycoprotein (MAG/siglec-4) and CD33 (siglec-3), thebinding of which to sialic acid was not previously known, also representmembers of the siglec family. Six other human siglec molecules (siglecs5-10) have been identified and characterised. These previously unknownmolecules exhibit a high degree of sequence similarity to CD33 in theirextracellular and intracellular domains and are collectively termed“siglecs standing in relation to CD33” (1; summary article). Reference(7) describes the cloning and characterisation of siglec-11 which isexpressed from human dendritic cells.

Table 1 gives an overview of the occurrence and potential functions ofthe previously described siglecs.

TABLE 1 Occurrence and potential functions of the previously describedsiglecs Name Occurrence Potential function Sialoadhesin MacrophageCellular interactions of (Sn, siglec-1) subpopulations macrophages CD22(siglec-2) B-lymphocytes Modulation of the B-cell dependent immuneresponse; homing in bone marrow CD33 (siglec-4) Myeloid precursorUnknown cells Myelin-associated Myelinated cells of the Receives themyelin glycoprotein (MAG, central and peripheral structure and function;siglec-4a): Schwann nervous system regulation of the neurite cellsmyelin protein (oligodendrocytes and growth (SMP, siglec-4b) Schwanncells) OB-BP2 (siglec-5) Neutrophile Unknown; presumably granulocytes,signal transduction monocytes OB-BP1 (siglec-6) Placenta (cyto- and Highaffinity leptin bond syncytiotrophoblasts); independent of Sia;B-lymphocytes function of the Sia bond unknown p75 / AIRM1 Naturalkiller cells Inhibitory receptor of (siglec-7) natural killer cellsSiglec-8 Eosinophilic Unknown; presumably granulocytes signaltransduction Siglec-9 Monocytes; Unknown; presumably neutrophilic signaltransduction granulocytes; CD16⁺ CD56⁻ cells Siglec-10 B-cells,monocytes Unknown; presumably and other leucocytes signal transductionSiglec-11 Dendritic cells, Cellular recognition; monocytes and othersignal transduction leukocytes

As can be seen from the table, siglecs also participate in the reductionof the immune response, the maintenance of the organisation of myelin inthe nervous system and in the hematopoesis.

The interactions mediated by siglecs may be of two types. Firstly,signals in the cells which express the corresponding siglec can beproduced by binding of the siglec molecules (e.g. damping of the B-celldependent immune response, inhibition of the cytotoxic activity ofnatural killer cells (NK cells) by phosphorylation of ITIM motives), orsignals can be produced at the cell bound by the siglec (e.g. regulationof the neurite growth of neurons by siglec-4a, [MAG] (1, 2)).

The effect of the binding partners of the siglecs can occur here in twoways (3). Firstly, monovalent substances can impair the biologicallyrelevant cross-linking of the siglec molecules to each other or impairit with other molecules. This would lead to a reduction of the signal.Secondly, polyvalent substances can reinforce the triggered signal.Consequently, a regulation in both directions is possible. Suitablespecific substances are required for these processes.

Sialic acid is a generic term for a large family of 9-carbon atom sugarswhich represent all the derivatives of neuraminic acid (Neu) orketo-desoxy-nonulsonic acid (KDN). Typically these are found on theexposed non-reducing ends of the oligosaccharide chains which are linkedto a large number of proteins and lipids.

The sialic acid binding site of the siglec lies in the N-terminal domainwhich is a V-set domain and which contains characteristic structuralfeatures for siglecs. The position of the binding site and the aminoacids participating in the binding were found by X-ray structuralanalysis of co-crystals of siglec-1 (sialoadhesin) and2,3-sialyllactose. The contributions of the functional groups of sialicacid to the binding were determined from Hapten inhibition experimentswith synthetic sialic acid derivatives (5, 6). Summarizing, thesestudies have shown that, among other aspects, the hydroxyl group on theC-9 of the sialic acid provides a substantial contribution as a hydrogendonor in a hydrogen bridge for binding and can be substituted by anamino group (5).

To regulate the biological functions mediated by siglecs, bindingpartners are required which occupy the binding sites with high affinity.

It is therefore the object of this invention to provide siglecinhibitors with a high affinity. A preferred object is the provision ofsiglec inhibitors with increased affinity which bind as specifically aspossible to single siglec receptors.

The object of this invention is solved by the provision of siglecinhibitors with the formula:

whereby

-   -   X signifies a negatively charged group such as a carboxy,        phosphate or sulphate group or a derivative of them;    -   Y signifies an H atom, an alkyl or aryl group, a hydroxy group,        a glycan, a polymer carrier molecule or a derivative of them;    -   Z is selected from O, N, C and S;    -   R1 signifies an H atom, a hydroxy group or a derivative of them;    -   R2 signifies a hydroxy or amino group or a derivative of them;    -   R3 signifies a hydroxy group or a derivative of it;    -   R4 signifies a hydroxy group or a derivative of it;    -   R5 signifies a substituted or unsubstituted amino group, whereby        the substituent is selected from    -   a substituted or unsubstituted formyl, alkanoyl, cycloalkanoyl,        aryl-carbonyl, heteroaryl-carbonyl, alkyl, aryl, cycloalkyl or        heteroaryl group, whereby these residues can also include one or        more unsaturated bonds,    -   whereby R4 acts as H acceptor and R5 as H donor;    -   R6 signifies an H atom or an alkyl group, a charged group or a        derivative of them;    -   R6′ signifies an H atom or an alkyl group, a charged group or a        derivative of them, whereby at least one substituent is selected        from R6 and R6′ is a hydrophobic group, preferably an H atom or        a methyl group; and    -   R7 signifies an H atom or any group, preferably a group for        improving the pharmacological properties of the siglec        inhibitor.

In the following some terms are defined as they are taken in relation tothis application.

The term “siglec” includes all siglec molecules. Reference should bemade to (8) for the definition of a siglec molecule. The amino acidsequences of the siglec molecules siglec-1 to siglec-10 can, forexample, be taken from the references listed in (1, Table 1). The aminoacid sequence of siglec-11 can be taken from (7). The amino acidsequences of the siglec molecules can also be obtained from the publiclyaccessible data base, Entrez (internet address:www.ncbi.nlm.nih.gov./entrez). The siglec molecules can in this respectbe present in their natural environment on naturally occurring cells orin artificial environments.

The term “siglec inhibitor” generally signifies the capability of acompound to inhibit the binding of a sialic acid molecule, in particularthe natural ligand, to the siglec protein. A siglec inhibitor accordingto the invention activates or deactivates a given siglec proteindepending on its structure. Preferably, the reference compound of thesialic acid is methly-α-5′-N-acetyl-neuraminic acid. Preferably, theinhibition can be determined by a Hapten inhibition test. The Hapteninhibition test is here based on Fc-chimeras which consist of N-terminaldomains of siglecs and the Fc part of the human IgG, complexed withradioactively marked anti-Fc antibodies and incubated with variousconcentrations of the potential inhibitors to be investigated, beforesuitable target cells, preferably human erythrocytes, are added. Afterovernight incubation at 4° C. the unbound complexes are removed bywashing the cells and the bound radioactivity is determined. From thedata obtained in this way, the concentrations are found which lead to50% inhibition of the binding (IC50 values) (5, 6). Specially preferredduring the Hapten inhibition test was 10 μl of the complex solution withan activity of 10³ Bq ¹²⁵I mixed with the same volume of a solution ofthe substance to be investigated (concentrated three times) andincubated at 4° C. for one hour. Then followed the addition of 10 μl ofa suspension of target cells, preferably 0.25 to 0.5% of humanerythrocytes and incubation overnight at 4° C. The unbound radioactivityis removed by washing the cells five times with 200 μl of washingbuffer, preferably phosphate-buffered saline with 0.1% (W/V) bovineserum albumin, and the cell-bound activity is determined with aγ-counter. As a control, preferably the binding of sialidase-treatedcells and of untreated cells is measured without the substance to beinvestigated. The inhibition is determined in that the value withoutinhibitor is set to 0% and that with the sialidase-treated cells is setequal to 100%.

The term “derivative” generally means in relation to the residues X, Y,R1-R4, that the group occurring in the neuraminic acid is substituted bya bioisosteric group, which exhibits essentially the same biologicalactivity. The concept of bioisosterics is known to the specialist.

The expression “a substituted formyl, alkanoyl, cycloalkanoyl,aryl-carbonyl, heteroaryl-carbonyl, alkyl, cycloalkyl or heteroarylgroup” means that the relevant groups exhibit substituents which leavethe biological properties essentially unmodified. This includes, forexample, low-alkyl substituents, such as for example methyl, ethyl,propyl and butyl groups.

According to the invention, X signifies a negatively charged group. Thisnegatively charged group presumably forms a salt bridge with an arginineresidue of the siglec receptor. The naturally occurring substituent is acarboxy group. Suitable derivatives of it are, for example, a phosphateor sulphate group. Furthermore, phosphonate and sulphonate groups arealso taken into account. Other suitable derivatives are a carboxymethylene or carboxy ethylene group.

According to the invention, Y signifies an H atom, an alkyl or arylgroup, a hydroxy group, a glycan, a polymer carrier molecule or aderivative of them. The naturally occurring substituent is a hydroxygroup. Suitable derivatives of the hydroxy group are in this respect anamino or thio group. Suitable glycans are in this respect hexoses,hexosamines and/or pentoses or derivatives of them, preferably glucoseor galactose or derivatives of them. Furthermore suitable glycans areoligo and polysaccharides, whereby the oligo and polysaccharides can beformed from a monomer or various monomers (mixed sugars). As polymercarrier molecules in this respect, carrier molecules are suitable whichimprove the pharmacological properties, such as longer retention time.Polymer carrier molecules with many bound siglec inhibitor ligandsenable cross-linking and consequently the activation of the siglecreceptor molecules. The application of such polymer carrier moleculestherefore enables fine regulation.

The carrier molecules preferably contain a core and various quantitiesof substances according to the invention which are bound to it throughsuitable spacers. The desired pharmacological effect can be controlledthrough the composition of the polymers comprising the core andsubstances according to the invention. Preferably, the polymers (core)are dendrimers, polyacrylamide or polylactide. The substances accordingto the invention for example, can be coupled either chemically orenzymatically to the polymers.

According to the invention Z signifies an atom selected from O, N, C orS.

According to the invention R1 is an H atom, a hydroxy group or aderivative of them. Suitable derivatives of the hydroxy group are inthis respect an amino or thio group which can be substituted asapplicable.

According to the invention R2 signifies a hydroxy or amine group or aderivative of them. The naturally occurring substituent is anamino-acetyl group. Suitable derivatives are, for example, those withwhich the amino group is substituted by an acetyl, propionyl, butyl orpentyl group. The alkanoyl group can in this respect furthermore besubstituted by one or more halogen atoms. Other suitable derivatives canbe taken from (Reference 5; compounds 4 to 12). The modification onposition R2 can contribute to an increase in the specificity of thesiglec inhibitor for a given siglec molecule.

According to the invention R3 signifies a hydroxy group or a derivativeof it. In this respect, suitable derivatives are, for example, an aminoor thio group which can be substituted where applicable.

According to the invention R4 signifies a hydroxy group or a derivativeof it. In this respect, suitable derivatives are groups which act as Hacceptors. Examples of derivatives in this respect are an amino or thiogroup, which can be substituted where applicable, whereby the H acceptorproperty is retained.

According to the invention R6 and R6′ signify independently of oneanother an H atom or an alkyl group, a charged group or a derivative ofthem, whereby at least one substituent is selected from R6 and R6′ is ahydrophobic group, preferably an H atom or a methyl group. Suitablederivatives are low alkyl substituents such as methyl, ethyl, propyl orbutyl groups. Suitable charged groups are, for example, carboxy,sulphate or phosphate groups.

According to the invention R7 signifies an H atom or any group,preferably a group for improving the pharmacological properties of thesiglec inhibitor. Groups for improving the pharmacological propertiescan be polymer carrier molecules. The complete siglec inhibitor shouldpreferably exhibit such a hydrophilicity which leads to an evendistribution of the inhibitor in a hydrophilic and hydrophobic phase.

Furthermore, the covalent or non-covalent binding of the siglecinhibitors according to the invention on natural glyco-conjugates andglyco-proteins is taken into consideration.

It was surprisingly found that through the introduction of hydrophobicsubstituents on the amino group of the 9-amino-9-desoxy-sialic acid, inparticular neuraminic acid, siglec inhibitors with increased affinityrelative to the reference compound 5′-acetyl-neuraminic acid wereobtained. According to preferred embodiments, siglec inhibitors areprovided which specifically bind to certain siglec proteins and inhibitthem.

According to a preferred embodiment, the alkanoyl group is selected froman ethanoyl, propanoyl, butanoyl, pentanoyl, hexanoyl, heptanoyl,octanoyl, nonanoyl and decanoyl group, preferably hexanoyl. According tothe invention branched alkanoyl groups are also taken intoconsideration.

In a further preferred embodiment the cycloalkanoyl group is selectedfrom a C₃ to C₆ cycloalkanoyl group, preferably cyclohexanoyl.

In another preferred embodiment, the aryl-carbonyl group is selectedfrom a C₄ to C₁₅ aryl-carbonyl group, preferably from a benzoyl group,naphthoyl group, anthracen-carbonyl group, whereby this residueprimarily takes part in the selectivity. The selectivity can be in thisway controlled through a suitable selection.

In another preferred embodiment the heteroaryl-carbonyl group isselected from a pyridyl-carbonyl group, chinaldine-carbonyl andthiophenyl-carbonyl group.

In another embodiment the alkyl group is selected from a C₁ to C₂₀ alkylgroup, preferably from a methyl, ethyl, propyl, butyl, pentyl and hexylgroup.

According to the invention branched alkyl groups are also considered.

In another preferred embodiment the cycloalkyl group is selected from aC₃-C₆ alkyl group.

In a further preferred embodiment the aryl group is selected from aphenyl, naphthyl, biphenyl and anthracen group. According to theinvention the aryl group can be selected both from condensed as well asnon-condensed aryl groups.

The heteroaryl group selected from a pyridyl, chinaldine and thiophenylgroup is also preferred.

The heteroaryl group comprises, according to the invention, bothcondensed and also non-condensed hetero-aromatic systems which are knownto the specialist.

A particularly preferred compound ismethyl-α-9-N-(naphthyl-2-carbonyl)-amino-9-desoxy-Neu5Ac. This sialicacid derivative binds approximately twelve times stronger to siglec-1than the reference compound 2-alpha-methyl-5-N-acetyl-neuraminic acidand stronger still to siglec-4a (approx. 236 times stronger).

Methyl-α-9-N-(biphenyl-4-carbonyl)-amino-9-desoxy-Neu5Ac is alsoparticularly preferred. This compound binds approximately 150 timesstronger to siglec-2 than the reference compound2-alpha-methyl-5-N-acetyl-neuraminic acid.

Furthermore, methyl-α-9-N-benzoyl-amino-9-desoxy-Neu5Ac is particularlypreferred. This compound binds approximately 704 times stronger tosiglec-4a (MAG) than the reference compound2-alpha-methyl-5-N-acetyl-neuraminic acid.

According to a preferred embodiment of the invention a siglec inhibitoris provided, whereby

-   -   X signifies a carboxy group, which should be present in an axial        position;    -   Y signifies an H atom, an O-methyl, O-benzyl group or a        derivative of a hydroxy group;    -   Z signifies an O atom;    -   R1 signifies a hydroxy group;    -   R2 signifies an amino-acetyl group;    -   R3 signifies a hydroxy group;    -   R4 signifies a hydroxy group;    -   R6 signifies an H atom;    -   R6′ signifies an H atom; and    -   R7 signifies an H atom.

With the exception of Y, the quoted substituents in this respect matchthe naturally occurring substituents of sialic acid.

The invention also provides a method for the manufacture of siglecinhibitors with increased affinity for a siglec molecule, which includesthe steps:

-   -   a) introduction of a substituent selected from the residues        according to the invention in the position R5 of neuraminic acid        or derivatives of it;    -   b) determination of the affinity of the product according to a)        for a siglec molecule;    -   c) selection of the products with increased affinity;    -   d) where applicable, further substitution of the selected        product according to c) in positions different from position R5,        preferably in position R2.

According to the invention, it was found that siglec inhibitors withincreased affinity for a siglec molecule can be obtained by introducinga hydrophobic substituent in position R5 of the neuraminic acid orderivatives of it. The determination of the affinity of the product fora given siglec molecule can in this respect occur by a binding assay ora Hapten inhibition test. The conditions for the Hapten inhibition assayare in this respect, as stated above, preferably as specified in (5, 6).The affinity of the selected products for a given siglec molecule can befurther increased by the introduction of substituents in positionsdifferent from position R5, preferably position R2. Suitablesubstituents for R2 are in this respect the substituents stated for R5.

The invention also provides a method for increasing the bindingselectivity of siglec inhibitors which includes the step of introducinga substituent selected from the residues for R5 according to the productof the present invention in position R5 of neuraminic acid orderivatives of it.

The synthesis of the compounds according to the invention is inprinciple possible for the specialist based on his general specialistknowledge. Preferred as the starting product is 5-N-acetyl-neuraminicacid, from which first the appropriate alkyl, aryl, alkyl-alpha-O- oralpha-S-glycosides are produced in a reaction sequence comprisingseveral steps.

The next step consists of replacing the hydroxy group on C9 (R5according to this invention) of the appropriate O- or S-glycoside of the5-N-acetyl-neuraminic acid by an amino group. This transformation can becarried out via the appropriate 9-O-tosyl compound. Preferably, thisreaction can be carried out using a modified Mitsunobu reaction. Thethus obtained alkyl, aryl, aralkyl-alpha-O- or alpha-S-glycosides of the9-amino-9-desoxy-5-N-acetyl-neuraminic acid finally supply theappropriate alkyl, aryl, alkyl-alpha-O- or alpha-S-glycosides of the5-N-acetyl-9-(biphenyl-4-carbonyl)-amino-9-desoxy-neuraminic acid andsimilar substances with a varying acyl residue on the C-9 (R5 accordingto the invention) constant amino group. The execution of this linkage ofacid function with amino group can be carried out in various ways, forexample by using the respective acid chloride or anhydride or with theaid of the carbodiimide method or via the method of the respective acidfunction, activated for example with nitrophenol, pentafluorphenol, etc.

This invention also provides pharmaceutical compositions comprising atleast one siglec inhibitor according to the invention and apharmaceutically compatible carrier. According to a preferred embodimentthe therapeutically applicable siglec inhibitors are as selective aspossible for a siglec molecule. Pharmaceutically compatible carriers areknown to the specialist.

These also include suitable dilution agents. Generally, any form ofadministering is suitable, e.g. intravenously, intraperitoneally,subcutaneously, intradermally, orally or topically, whereby the oraladministration is preferred in this respect.

The amount of medicament to be administered can be determined routinelyby the doctor.

Furthermore, this invention provides the application of the siglecinhibitors according to the invention for the treatment of diseasesmediated by siglecs, preferably diseases of the immune system. Siglec-2participates in the regulation of the immune response dependent onB-cells. This invention therefore indicates the application of thesiglec inhibitors for the regulation of the immune response dependent onB-cells. In this respect, according to the invention, allergies,auto-immune diseases and chronic inflammations are quoted as subjectsfor a siglec inhibitor treatment.

Siglec-4a exhibits a neurite growth-inhibiting effect. The siglecinhibitors according to the invention are therefore suitable forneutralizing the neurite growth inhibiting effect of siglec-4a andtherefore possess the capability of improving the regenerationcapability of damaged nerves, for example in the treatment ofparaplegia. Siglec-7 takes part, for example, in the regulation of thecytotoxic activity of NK cells. The sialic acid derivatives according tothe invention are therefore suitable for the regulation of the cytotoxicactivity of these cells. For example, treatable diseases in this regardare cancer diseases and viral diseases, in particular AIDS.

There are indications of the participation of other siglecs in thecontrol of the immune system, cf. Table 1. The siglec inhibitorsaccording to the invention are therefore also suitable for the controlof the immune system.

The preferred siglec inhibitors according to the invention result in anincreased immune response dependent on B-cells, which can be verified inparticular by an increased Ca²⁺ secretion. This increased Ca²⁺ secretionarises through the application of the preferred siglec inhibitorsaccording to the invention, verifiable, for example, in tests with Daudicells or B-cells from mice. This increased immune response, dependent onB-cells, induced through the application of the preferred siglecinhibitors of this invention, opens up many promising possibilities forthe manufacture of medicaments for the treatment of diseases associatedwith immune defects. A compound preferred in this connection ismethyl-α-9-N-(biphenyl-4-carbonoyl)-amino-9-desoxy-Neu5Ac (illustratedin the examples). This compound also shows in particular a veryremarkable selective affinity for hCD22. Medical indications for whichthe preferred inhibitors show special potential are diseases in whichthe immune response within the scope of the B-cell activation isdisturbed. Examples of this are the Common Variable Immunodeficiency(CVID) and the IgA deficiency. CVID patients have B-cells which howevercannot initiate an effective immune response and are characterised by ahypogammaglobulinemia. They suffer from severe infectious diseases andcan currently only be treated with immune globulins, which however is aproblematic therapy with regard to the significant risks and restrictedscope of application. Patients with IgA deficiency could also be treatedwith immune globulins, which though is often not undertaken due to therisks described above and also because these patients often only exhibitslight symptoms.

In the following, examples are described which explain the invention,but which should not restrict it. When making use of the invention,other applications will open up for the specialist which are alsoincluded according to the invention.

Materials and Methods

Synthesis of Siglec Inhibitors

As an example of the production of the above quoted substances acylatedon the amino group of the methyl-α-5-N-acetyl-9-amino-9-desoxyneuraminic acid, the synthesis of themethyl-α-5-N-acetyl-9-N-(biphenyl-4-carbonyl)-amino-9-desoxy-neuraminicacid (3) is described here.

Methyl-α-5-N-acetyl-9-azido-9-desoxy-neuraminic acid (1)

-   -   a) From methyl-α-5-N-acetyl-9-O-tosyl-neuraminic acid methyl        ester, according to methods known from publications, via the        appropriate 9-azide and saponification of the ester group.    -   b) Direct from known methyl-α-N-acetyl-neuraminic acid through        chemical reaction based on the Mitsunobu reaction.

A solution of the triethyl-ammonium salt of themethyl-α-5-N-acetyl-neuraminic acid (0.1 g) in dry pyridine (0.4 ml) andN,N-dimethyl-formamide (DMF) (1 ml) is concentrated in a vacuum and thenfumed with dried DMF 1-2 times (A). Tetramethylguanidine (0.19 g) isdissolved in a mixture of dry pyridine (0.25 ml) and DMF (1 ml), thesolution is concentrated in a vacuum and then fumed with dry DMF 1-2times (B). A is dissolved in DMF (0.8 ml), the solution added to B and98-100% formic acid (0.13 ml) is added. The solution of A+B is added toa mixture of triphenylphosphine (0.17 g) and diisopropylazodicarboxylate(0.125 ml) in dried tetrahydrofurane (1.2 ml), which first was incubatedat 0° C. for about 15 minutes. After about 24 hours at 0° C.→20° C. thereaction is terminated and about 0.5 ml of methanol are then added. Thesolution is concentrated in a vacuum, shaken out with ethyl acetate/H₂Oor methylene chloride/H₂O and chromatographed on silica gel (Flashmethod).

Elution: First methanol/ethyl acetate/acetic acid (20%) 1/6/1, then1/4/1.

Yield: 70-80% of theory.

Methyl-α-5-N-acetyl-9-amino-9-desoxy-neuraminic acid (2)

The 9-azido compound (1) is hydrogenated with palladium oxide in H₂O atnormal pressure.

Yield: 95%.

Methyl-α-5-N-acetyl-9-N(biphenyl-4-carbonyl)-amino-9-desoxy-neuraminicacid (3)

Chemical reaction of biphenyl-4-carboxylic acid (0.4 g) with4-nitrophenol (0.28 g) in ethyl-acetate in the presence ofN,N′-dicyclohexyl-carbodiimide (0.416 g) at room temperature yieldsrespective biphenyl (4)-carboxylic acid 4-nitrophenylester, whichcrystallises from ethyl acetate/diethyl ester/hexane.

Methyl-α-5-N-acetyl-9-amino-9-desoxy-neuraminic acid (2) (30 mg)dissolved in dried DMF (0.6 ml) reacts in the presence of triethylamine(12.9 ml) completely with the above quoted nitrophenylester (39 mg) atroom temperature. Purification occurs through chromatography on silicagel (Flash method).

Elution: First methanol/ethyl acetate/acetic acid (20%) 1/6/1, then1/5/1, finally 1/4/1.

Yield of (3): 93%.

High resolution NMR spectroscopy and FAB-MS show the structure of thesynthesised products.

Hapten Inhibition Assay

The Hapten inhibition assay is carried out under the conditions quotedin (5, 6).

Results

TABLE 2 Siglec- Siglec-1 Siglec-2 Siglec-2 4a (sialo- (human (murine(MAG) adhesin) CD22) CD22) Structure IC50 rIP IC50 rIP IC50 rIP IC50 rIPMethyl-α-Neu5Ac 4716 1.0 884 1.0 1388 1.0 4689 1.0 Methylthio-α-Neu5Ac2775 1.3 600 0.8 1367 1.6 4250 1.1 Benzylthio-α-Neu5Ac 2000 3.0 280 4.1n. d. n. d. n. d. n. d. 2,3-sialyllactose 1600 7.3 385 2.4 n. d. n. d.n. d. n. d. Methyl-α-5-N-glycolyl-neuraminic acid n. d. n. d. n. d. n.d. 1013 1.0 703 8.3 Methyl-α-methyl-α-5-N-fluoracetyl- 280 17.1 625 1.9n. d. n. d. n. d. n. d. neuraminic acid Methyl-α-methyl-α-5-N- >>10 mMn. a. 2500 0.4 n. d. n. d. n. d. n. d. fluoracetylaminoacetyl-neuraminicacid (90%) Benzyl-α-5-N-propanoyl-neuraminic acid 2500 4.8 215 3.4 n. d.n. d. n. d. n. d. Benzyl-α-5-N-butanoyl-neuraminic acid 5300 1.5 250 2.7n. d. n. d. n. d. n. d. Benzyl-α-5-N-benzoyl-neuraminic acid <10mM >0.27 >10 mM <0.05 n. d. n. d. n. d. n. d. (27%) (63%)Methyl-α-ketodesoxy-nonulsonic acid (KDN) >>1.0 mM <<0.5 >1 mM <0.8 >>1mM <<2 >2 mM <1.5 (225%) (74%) (117%) (74%)Methyl-α-5-N-(etoxycylcobutendion)- 4300 0.5 >>5 mM <<0.15 940 2.3 33671.5 neuraminic acid (92%) Methyl-α-9-methylsulphoxido-9-desoxy- 697 8.52000 <0.05 n. d. n. d. n. d. n. d. Neu5AcMethyl-α-9-methylthio-9-desoxy-Neu5Ac >10 mM <<0.27 5000 <<0.05 n. d. n.d. n. d. n. d. (116%) Methyl-α-9-methylsulphonyl-9-desoxy- 3400 0.1 2000<<0.05 n. d. n. d. n. d. n. d. Neu5AcMethyl-α-9-N-aminoacetyl-9-desoxy-Neu5Ac 3400 3.3 7480 0.3 n. d. n. d.n. d. n. d. Methyl-α-9-N-oxamido-amino-9-desoxy- n. d. n. d. >4000 <0.2n. d. n. d. n. d. n. d. Neu5Ac (57%) Methyl-α-9-carboxy-Neu5Ac >>1000<<2.4 >4000 <0.2 n. d. n. d. n. d. n. d. (87%) (63%)Methyl-α-9-N-acetyl-amino-9-desoxy- 2150 3.7 3817 0.2 1800 0.6 1.750 2.7Neu5Ac Methyl-α-9-N-trifluoracetyl-amino-9-desoxy- 550 10 4000 0.5 n. d.n. d. n. d. n. d. Neu5Ac Methyl-α-9-N-thioacetyl-amino-9-desoxy- 733 6.56000 0.3 n. d. n. d. n. d. n. d. Neu5AcMethyl-α-9-N-acetoacetyl-amino-9-desoxy- 1007 6.2 >10 mM <0.05 n. d. n.d. n. d. n. d. Neu5Ac (62%) Methyl-α-9-N-hexanoyl-amino-9-desoxy- 198 541458 0.7 n. d. n. d. n. d. n. d. Neu5AcMethyl-α-9-N-cyclohexanoyl-amino-9-desoxy- 110 50 >>1 mM 0.2 383 4.1 7606.9 Neu5Ac (93%) Methyl-α-9-N-(2,3-diacetylaminopropanoyl)- 25002.3 >>10 mM <<0.1 n. d. n. d. n. d. n. d. amino-9-desoxy-Neu5Ac (80%)Methyl-α-9-N-(gly)₂-amino-9-desoxy-Neu5Ac 5000 0.9 >>10 mM <<0.1 n. d.n. d. n. d. n. d. (100%) Methyl-α-9-N-(gly)₃-amino-9-desoxy-Neu5Ac >10mM <<0.27 <<10 mM >>0.05 n. d. n. d. n. d. n. d. (88%) (11%)Methyl-α-9-N-(ethoxycyclobutendion)-amino- 240 32 1333 0.2 n. d. n. d.n. d. n. d. 9-desoxy-Neu5Ac Methyl-α-9-N-(pyridine-3-carbonyl)-amino-9-25 170 4000 0.5 445 2.3 335 14 desoxy-Neu5AcMethyl-α-9-N-benzoyl-amino-9-desoxy- 7 704 539 1.8 290 5.7 1094 3.7Neu5Ac Methyl-α-9-N-(3,5-dihydroxybenzoyl)-amino- 28 166 525 1.7 73 191467 n. d. 9-desoxy-Neu5Ac Methyl-α-9-N-(acetamidobenzoyl)-amino-9- 6277 295 3.0 580 2.4 1033 n. d. desoxy-Neu5AcMethyl-α-9-N-(phenylacetyl)-amino-9-desoxy- 367 7 n. d. n. d. n. d. n.d. n. d. n. d. Neu5Ac Methyl-α-9-N-(2,4-dinitrobenzoyl)-amino-9- 205 18733 0.3 287 7.7 593 7.9 desoxy-Neu5AcMethyl-α-9-N-(4-methoxybenzoyl)-amino-9- 12 393 42 8.4 120 19 667 7.4desoxy-Neu5Ac Methyl-α-9-N-(pentafluorbenzoyl)-amino-9- 107 42 7000 0.3360 3.3 2500 2.0 desoxy-Neu5AcMethyl-α-9-N-(biphenyl-4-carbonyl)-amino-9- 22 218 52 13 4 150 1220 5.0desoxy-Neu5Ac Methyl-α-9-N-(biphenyl-4-acetyl)-amino-9- n. d. n. d. 30000.3 35 29 123 48 desoxy-Neu5AcMethyl-α-9-N-(biphenyl-2-carbonyl)-amino-9- n. d. n. d. 2600 0.3 97 10647 9.5 desoxy-Neu5Ac Methyl-α-9-N-(phenoxy-3-benzoyl)-amino-9- n. d. n.d. 540 1.5 10 111 887 6.7 desoxy-Neu5Ac Methyl-α-9-N-(diphenylacetyl)-amino-9- 150 31 4400 0.2 35 n. d. 103 n. d. desoxy-Neu5AcMethyl-α-9-N-(naphthyl-2-carbonyl)-amino-9- 20 236 78 12 6 167 270 18desoxy-Neu5Ac Methyl-α-9-N-(naphthyl-1-carbonyl)-amino-9- 56 84 3000 0.337 27 92 64 desoxy-Neu5Ac Methyl-α-9-N-(naphthyl-2-acetyl)-amino-9- 36713 1750 0.5 8 131 71 83 desoxy-Neu5AcMethyl-α-9-N-(anthracen-5-carbonyl)-amino- 162 28 750 0.5 338 4.9 6477.4 9-desoxy-Neu5Ac Methyl-α-9-N-(cyclobutendion)-amino-9- n. d. n. d.n. d. n. d. 180 5.7 620 7.4 desoxy-Neu5AcMethyl-α-9-N-(chinaldine-2-carbonyl)-amino- 87 54 >>2.5 mM <<0.2 41 n.d. >>10 mM n. d. 9-desoxy-Neu5Ac (100%) (100%)Methyl-α-9-N-dansyl-amino-9-desoxy-Neu5Ac 317 19 260 2.6 n. d. n. d. n.d. n. d. Methyl-α-9-N-fluoresceinyl-amino-9-desoxy- 106 41 >>1.5 mM<<0.5 77 28 100 48 Neu5Ac (121%) gly = glycine

The IC50 value is the siglec inhibitor concentration which leads to 50%inhibition in the binding in the Hapten inhibition assay. The rIP valueof each sialic acid derivative was determined by forming the quotient ofthe IC50 value of the comparative compound 5-N-acetyl-neuraminic acidand of the IC50 value of the compound to be investigated. Sialic acidderivatives with an rIP value of >1.0 therefore bind better than thereference compound and an rIP value of <1 shows that the compound bindsworse to the receptor than the reference compound. n.d. signifies thatno determination had been conducted.

The compound BPC-Neu5Ac (shown below) was used according to knownmethods in test series for the investigation of the selectivity and theactivity. The stimulation of Daudi cells with anti-IgM in the presenceof BPC-Neu5Ac produced a rise in the Ca²⁺ concentration. The applicationof this compound also led to a clearly increased Ca²⁺ concentration inprimary B-lymphocytes from human blood which were stimulated withanti-IgM. This data indicates that the increased Ca²⁺ signal of thetreated cells is caused by a specific inhibition of the ligand bindingdomain of CD22. This impairment with regard to the binding of ligandsleads to an incomplete activation of the intracellular inhibitor domainof CD22.

Methyl-α-9-N-(biphenyl-4-carbonyl)-amino-9-desoxy-Neu5Ac (BPC-Neu5Ac)REFERENCES

-   1) Crocker et al., Immunology 102 (2001), 1-14.-   2) Crocker et al., (2000) The siglec family of I-type lectins in    Carbohydrates in Chemistry and Biology, Vol. 4, B. Ernst et al.,    publisher, Wiley-VCH, pp. 579-595.-   3) Kelm, S. (2001), Ligands for siglecs. In Mammalian Carbohydrate    Recognition Systems, P. R. Crocker, publisher (Berlin, Springer),    pp. 153-176.-   4) May et al., Mol. Cell 1 (1998), 719-728.-   5) Kelm et al., Eur. J. Biochem. 255 (1998), 663-672.-   6) Strange et al., Eur. J. Biochem. 258 (1998), 677-685.-   7) Li, N. et al., Cloning and Characterization of Siglec-11, a novel    sialic acid binding member of the Ig Superfamily from human    dendritic cells; J. Biol. Chemistry 2001 (in printing).-   8) Crocker et al., Glycobiology 8 (1998), v.

1. A method of increasing the binding selectivity of siglec inhibitorscomprising introducing at the C-9 position of neuraminic acid asubstituted or unsubstituted amino group, whereby the substituent isselected from the group consisting of substituted or unsubstitutedformyl, alkanoyl, cycloalkanoyl, aryl-carbonyl, alkyl, aryl, andcycloalkyl groups, wherein these groups can also include one or moreunsaturated bonds, and wherein the neuraminic acid has the followingformula:


2. A method for producing siglec inhibitors with increased affinity fora siglec molecule comprising: a) replacing the hydroxyl group at the C-9position of neuraminic acid with a substituted or unsubstituted aminogroup, whereby the substituent is selected from the group consisting ofsubstituted or unsubstituted formyl, alkanoyl, cycloalkanoyl,aryl-carbonyl, alkyl, aryl, and cycloalkyl groups, wherein these groupscan also include one or more unsaturated bonds; b) determining theaffinity of the product according to a) for a siglec molecule; c)selecting the products with increased affinity; and d) optionally,further substituting the amino group at position C-5 of the product ofstep c).